One of the ways that genetic variability is maintained within a population is through recombination, a process involving the exchange of genetic information among different DNA molecules that results in a reshuffling of genes. To provide recombination in the field of genetic engineering, a vector is usually used. The most commonly used vector is the DNA plasmid, a small genetic element that permits microorganisms to store additional genetic information.
Plasmids are useful elements in many biotechnological applications these days. For example, in the medical and diagnostic fields, genetic engineering of cells is performed using plasmids that carry a gene encoding a protein, which is not expressed in the native cell.
Another use of plasmids as vectors is in the field of gene therapy, which is expected to be one of the fastest growing areas in the next decade. Gene therapy is a therapeutic strategy where nucleic acids are introduced to human cells to cure genetic defects e.g. cystic fibrosis. The first human gene therapy trials began in 1990, using an ex vivo strategy. In this approach, the patient cells are harvested and cultivated in the laboratory and then incubated with vectors to introduce the therapeutic genes. Even though approaches for delivering genes based on in vivo gene therapy, in which the virus is directly administered to the patients, have been suggested more recently as an alternative, the plasmid retains its importance in gene therapy.
Thus, the increased use of such biotechnological applications results in a need for large quantities of plasmid DNA. To this end, an efficient large-scale purification process, which can meet specifications in purity and quantitation, is required. Today, many purification methods are available for smaller molecules of sizes of about 10 nm, such as proteins. However, for the larger DNA plasmids, which are of sizes of 100 nm and above, much fewer purification methods are available.
Conventionally, the production of plasmid DNA involves fermentation, primary purification and high-resolution separation. Recently many methods have been suggested involving use of chromatography as the method for purification of plasmid DNA. However, the use of chromatography as a single purification technique alone for plasmid DNA involves several drawbacks, such as a slow diffusion, low capacity of the matrices, shearing of large plasmids and recovery of the plasmid in high salt concentration. Therefore, there is a need of a primary purification step before chromatography.
Use of a two-phase system has been suggested for purification of plasmid DNA. Aqueous two-phase systems are extremely mild and have shown a strong potential for use as a primary recovery step for plasmid purification. Plasmid DNA is today often produced in Escherichia coli and involves an alkaline lysis step for release of plasmid DNA from the bacterial cells. Several contaminants such as RNA, genomic DNA, proteins, cells and cells debris are released in the alkaline lysis step. Ribeiro et al. (S. C. Ribeiro et al: Isolation of Plasmid DNA from Cell Lysates by Aqueous Two-Phase Systems, 2002 Wiley Periodicals) has shown that plasmid DNA can be isolated in aqueous two-phase systems consisting of polyethylene glycol (PEG) and a salt. PEG is a linear polymer of ethylene oxide groups. The polymer is soluble in water, and at a certain salt concentration a two-phase system consisting of PEG and salt can be achieved. The PEG polymer can be removed by filtration or dialysis, which however often decreases the yield. Another drawback with this method is that PEG is a relatively expensive chemical, which will be of importance in large-scale processes. Furthermore, plasmid DNA resulting from this method will be present in an environment of high salt concentration, which is a disadvantage in applications such as gene therapy.
An alternative two-phase system differs from the PEG/salt systems, in the sense that the system is created by temperature-induced phase separation. More specifically, this means that a thermoseparating polymer is used, which polymer solution will separate into two phases when its temperature is increased to a point above its cloud point (CP). The above discussed PEG polymer can in fact be used as a thermoseparating polymer, but its high cloud point, which is 111.7° C. at a 10% solution in water, renders PEG systems highly unsuitable for separation of delicate biological materials. Thermoseparating two-phase systems have been suggested for partitioning of some proteins, such as enzymes, and for a water-soluble steroid.
More specifically, Harris et al. (P. A. Harris et al: Enzyme purification using temperature-induced phase formation, Bioseparation 2: 237-246, 1991) disclose the purification of the enzyme 3-phosphoglycerate kinase and hexokinase from a cell homogenate of baker's yeast in an aqueous two-phase system. The system used comprises a random copolymer of ethylene oxide (EO) and propylene oxide (PO) known as UCON 50-HB-5110 and dextran or hydroxypropyl starch. The EO-PO copolymer has a cloud point that is much lower than that of PEG, namely 50° C.
Further, Alred et al (Patricia A. Alred et al: Partitioning of ectdysteroids using temperature-induced phase separation, Journal of Chromatography, 628 (1993) 205-214) discloses a study of the partitioning of the ectdysteroids α-ecdysone and β-ecdysone in an aqueous two-phase system by thermoseparation. The system comprises the same components as the above-mentioned, namely the ethylene oxide-propylene oxide random copolymer UCON 50-HB-5100 and dextran. Due to the high levels of ecdysteroids recovered, 73.6% and 85.6%, respectively, such a system is suggested as an analytical or a preparative technique for ecdysteroids.
Finally, Persson et al (Persson et al: Purification of recombinant apolipoprotein A-1 expressed in Escherichia coli using aqueous two-phase extraction followed by temperature-induced phase separation, Journal of Chromatography B, 711 (1998) 97-109) describe a method of purification of recombinant apolipoprotein A1 in aqueous two-phase systems comprising ethylene oxide-propylene oxide random copolymers and hydroxypropyl starch. The polymer system is thermoseparating in the sense that it separates into one water-rich and one polymer-rich phase when heated above a critical point. It was shown that apolipoprotein could be partitioned to the top EO-PO copolymer phase while contaminating proteins and DNA was partitioned to the bottom phase.
In summary, there is still a need of improved methods for the purification of plasmid DNA from cell lysates.